Antimicrobial and cleansing composition

ABSTRACT

The invention relates to a antimicrobial and cleansing composition consisting of a. a polymeric biguanide, and b. a metal ion chelating agent, and g. a solvent, wherein the ratio of a:b is between 0.75:2.25 and 1.25:2.25, or about 1:2, and optionally one or more of the following ingredients; c. a quaternary ammonium salt, d. at least one surfactant, e.a humectant and f. a gelling agent. The invention further relates to said composition for use in a treatment, prevention or reduction of a disease, disorder or condition related to microbials, or treatment, prevention or reduction of infection, or inflammation, or infected wounds, or inflamed wounds, or treatment, prevention or reduction of biofilms, or removal of necrotic tissue. A liquid flow system may be used for administration of the composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/076,072, filed Aug. 7, 2018, which is a U.S. National Phase under 35U.S.C. § 371 of PCT/EP2017/052713, filed Feb. 8, 2017, and which claimspriority to Sweden Application No. 1650162-9, filed Feb. 9, 2016, theentire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention is directed toward an antimicrobial, cleansingcomposition and use thereof, in particular for therapeutic use inbiofilms, soft-tissue wounds, infected cavities, and infected bones.

BACKGROUND Infected Wound

Infections occur in a minor but significant portion of the patientsundergoing joint replacement surgery or fracture fixation, particularlythose with open fractures. Once established, infections are difficult toeradicate, especially in the case of bacterial biofilm formation onimplanted hardware. Wounds are a significant source of cost to patientsas well as to health care system, specially wound that are complicatedwith infection.

Biofilm formation on orthopaedic implants is attributed to theglycocalyx-mediated surface mode of bacterial growth and is usuallytreated through a secondary surgery involving irrigation, debridementand the appropriate use of antibiotics, or complete removal of theinfected implant.

When a wound is colonized with microorganisms they survive aspolymicrobial communities encased within a matrix of extracellularpolymeric substance (EPS). This community of microorganisms are attachedto each other, often in conjunction with a surface, and form communityof microorganisms. The combination of a community of microorganisms,encased within self-generated EPS and attached onto a surface (liquid orsolid) is simply defined as a biofilm. In heavily contaminated, infectedor chronic wounds with bacteria colonization and biofilm, thedebridement is the first step to facilitating successful wound closure.Wound debridement of infected tissues may help control infection andbioburden.

Research has shown that the lowest concentration required to kill oreliminate bacterial biofilm for many antibiotics actually exceeds themaximum prescription levels for the antibiotics. The widespread use ofantibiotics has resulted in the development of numerous microbialresistances. The continuing rise of bacterial resistance to antibioticsis a serious concern. Therefore, there is an unmet need to develop andinclude parallel approaches that target infections, especially thosecomplicated by the presence of biofilms.

In general, biocides have a broader spectrum of activity thanantibiotics. Antibiotics tend to have specific intracellular targets,while biocides or antiseptics or antimicrobials (which terms areinterchangeable in this specification) may have multiple targets.

Antiseptics are effective through many mechanisms of action. This makesthe development of resistance to them unlikely, in contrast toantibiotics. Maintenance debridement and use of topical antimicrobials(antiseptics) are believed to be more effective than antibiotic therapy.

In recent years, topical antimicrobial agents have become the first lineof treatment in managing bacterial burden, particularly in chronicwounds. The use of many antiseptics in wound management must be subjectto a risk-benefit assessment of possible local toxicity and beneficialantibacterial action. In short, it is advised that, before use, thebeneficial anti- microbial effects and bioavailability should be weighedagainst any possible cellular toxicity.

A variety of antimicrobial compositions, articles and methods have beensuggested. However, such wound compositions and methods possess variousdeficiencies and shortcomings, and therefore also prevent long term useof the composition.

EP1404311 discloses a composition for use as a wound treatment agentwhich is suitable as a washing or shower gel for decontaminatingsurfaces of the body, for dissolving incrustations or scabs from bodysurfaces or as a dissolving gel for dressings and for changing moistdressing. Disclosed is a use of a surfactant and biguanidehydrochloride, and use of amphoteric or non-ionic surfactants incombination with biguanide hydrochloride (PHMB). Furthermore, thepreferred surfactant for this formulation is a betaine and, inparticular, an amidoalkyl betaine of a fatty acid. However, saidformulation does not contain any compound, which might enhance biocidaleffect of PHMB. On the contrary, said surfactants might reduce thebiocidal effect of PHMB, which may have negative effects on the furthertreatment of wounds.

EP2896395 discloses a liquid or a gel containing polyhexamethylenebiguanide (PHMB) and a non-ionic surfactant and at least oneco-surfactant with or without substances obtained from herbs, fortopical wound treatment. The preferred non-ionic surfactant aretri-block co-polymers of polyethylene oxide and polypropylene oxide. Thetri-block copolymer is preferably a poloxamer, particularly poloxamer188. Poloxamers generally are believed to be ineffective in woundhealing, but effective in reducing postsurgical adhesions in severaltest systems. Furthermore, there is no mention of the use of achelating/sequestrant agent in combination with biguanide hydrochloride.

US2012/0107415 discloses a combined disinfection and decontaminationagent comprising at least one vitamin, at least one metal ion, at leastone active-surface compound, and at least one further antimicrobialactive substance. The antimicrobial active substance is an alcoholicactive substance.

US2009/0069436 discloses an antimicrobial hand wash (skin) compositioncomprising biguanide or a quaternary compound. Also disclosed is acomposition for skin rub, in which case it may additionally comprise atleast one alcohol, at least one lipogel and at least one oil.Alternatively, the composition may be a skin wash, in which case it mayadditionally comprise at least one detergent agent and at least oneamphoteric surfactant.

US2002/0022660 discloses use of at least one alcohol (30-65 percent byweight) and biguanide and a combination of a surfactant as a deeppenetrating antimicrobial compositions for the skin as outlined below.The use of alcohols is preferably avoided, due to its known highcellular toxicity.

WO2007068938 discloses an antimicrobial composition suitable for use onskin and wounds comprising a source of an antimicrobial agent and anagent, which disrupts biofilms. Disclosed is a use of EDTA as the agentthat disrupts biofilms and the antimicrobial agent is selected from thegroup of iodine, ionic silver or an oxidizing agent, such as sodiumhypochlorite or chlorine dioxide. These antimicrobial agents have shownto have higher cellular toxicity.

WO2013086181 discloses a combination of at least one antimicrobialpolymeric biguanide and at least one antimicrobial vicinal diol todiminish or eliminate biofilm communities. Described is a method oftreating a surface, or a surgical dressing, and in dentistry, such asfor gingivitis. Since the antimicrobial monoalkyl vicinal diols in thisinvention have low or negligible water solubility, adding a surfactantis necessary. Suitable surfactants disclosed are cationic, anionic,non-ionic, amphoteric and ampholytic surfactants. However, thesesurfactants are used in high doses, particularly anionic surfactants innumerous forms and different applications, which results in a need oflarger amounts of antimicrobial agent, such as polymeric biguanide tomaintain the composition's biocidal effect. This raises concernregarding cellular toxicity of the composition. The cellular toxicity ofthe said composition has not been studied.

EP 1139759 discloses a disinfectant and cleaner solution that isformulated to apply on hard surface. The composition comprises a polymerbiguanide, a quaternary ammonium salt, a sequestrant, and a cleaningagent. It does not disclose a use of the composition in wound treatmentor on a vital surface. Therefore, the cellular toxicity of thecomposition is not tested.

A common treatment in both dentistry and the food/water industry is theuse of anti-biofilm agents, such as EDTA for controlling biofilms. EDTAcompositions are highly effective in eliminating existing biofilms, andpreventing biofilm formation. At low concentrations EDTA has been shownto prevent biofilms by inhibiting the adhesion of bacteria. Furthermore,it has also been shown to reduce biofilm colonization and proliferation.

Biguanide compound composed of a synthetic mixture of polymers. Theantiseptic agent polyhexamethylene biguanide (also known as polihexanideor PHMB) has been used for over 60 years in a wide range of applicationsfrom swimming pool sanitizers to preservatives in cosmetics and contactlens solutions. In recent years, PHMB has been used as a woundirrigation fluid. PHMB has an effect on both planktonic bacteria andthose in biofilms. It has been shown that PHMB is safe for clinical use.In the standard comparative tests of 12 the biocompatibility testsagainst other commonly used therapiesPHMB has demonstrated itssuperiority to chlorhexidine, povidone-iodine, triclosan, silver andsulphadiazine. In addition, no known resistance to PHMB has beenreported, most likely owing to its rapid and non-specific bactericidalactivity.

Ingredients commonly used in antibacterial composition, such aschlorhexidine, alcohols and glycols have undesired side effects.Especially for long term treatment, i.e. more than two days, theseingredients may cause side effects, such as cell toxicity.

US2013/0150451 discloses a composition comprising a biguande and avicinal diol (a monoalkyl glycol, monoalkyl glycerol or a monoacylglycerol). The composition may further comprise a chelating agent, asurfactant, etc.

Handler, SIAM 22, 18-21, April 2016, page 3, line 4-7, “Hydrophobic longchain alcohol”, discusses the toxicity of alcohol, such as vicinaldiols. Hall et. al, www.ajicjournal.org, Vol 37, No 4, page 326, leftcolumn, line 40-47, discloses other undesirable effect of alcohol-basedhand rubs including absorption into the blood and the removal of lipidconsidered essential for the integrity of the skin. See also SevcikovaPetra, et. Al., European J. of Lipid Science and Technology, April 2014,page 448, line 35-36 and 455, left column, line 21-28 showing that1-monoacylglycerol has a dose dependent cytotoxicity from mild tosevere.

Yi-Ching Li et. al, J. Dental Science (2014), 9, 130-135, discloses onpage 133 that chlorhexidine induces apoptosis at lower concentration andcauses necrosis at higher concentration on fibroblasts.

Animal and human studies have shown that Ethylene glycol alkyl ethers(EGAEs) can cause adverse reproductive, developmental, and hematologicaleffects through inhalation, dermal absorption, and ingestion (AndrzejStarek et. al, Arch Toxicol (2008) 82: 125-136, especially on page 126,line 8-11. On page 127, right column, line 20-21 they demonstrated alsohematological changes in the peripheral blood in their material thatwere produced by all of four tested EGAEs.

SUMMARY

The present invention relates to antimicrobial and cleansingcompositions, whereby the composition has minimal or no cellulartoxicity for mammal cells. The new composition is especially useful forlong term treatment of wounds, i.e. treatment for a period of more thantwo days, five days, a week or two weeks. The new composition reducescosts for health care.

The present invention relates to a composition consisting of

a. a polymeric biguanide,

b. ion chelating agent, and

g. a solvent

whereby the composition in addition optionally may consist of one ormore of the following ingredients

c. a quaternary ammonium salt,

d. at least one surfactant,

e. a humectant, and

f. a gelling agent.

In one embodiment, the composition does not consist an anionicsurfactant or an alcohol. In another embodiment, the composition has apH between 4 and 8. A buffer may be used in the composition. In afurther embodiment, the solvent is water.

The polymeric biguanide may be polyhexamethylene biguanide.

The ion chelating agent may be ethylenediaminetetraacetic acid (EDTA),as a disodium, trisodium, tetrasodium salt or mixtures thereof. In oneembodiment, the ion chelating agent is disodium EDTA. In anotherembodiment, the ion chelating agent is trisodium EDTA. In a furtherembodiment, the ion chelating agent is tetrasodium EDTA. In oneembodiment, the ion chelating agent is a combination of disodiumethylenediaminetetraacetic acid salt and trisodiumethylenediaminetetraacetic acid salt.

The present composition regards a non-alcoholic antimicrobialcomposition, which is an effective antimicrobial, cleansing productswith no or negligible cellular toxicity. The present innovation providesan antimicrobial, cleansing composition which combines known andeffective biocide agents in low dose. It is believed that due tosynergistic interaction, said composition exhibits a total antimicrobialeffect that is greater than the sum of effects from all individualantimicrobial agents. The result from an experimental animal study, showthat said compositions with multiple low dose biocides agents result ineffective antimicrobial, cleansing products with no or negligiblecellular toxicity and comparable with medical honey. The new compositionis especially useful for long term treatment, over a period of one, twoor three weeks, of a wound that may be infected or inflamed.

In one embodiment, the composition consists of

a. a polymeric biguanide, and

b. a metal ion chelating agent, and

g. a solvent,

wherein the ratio of a:b is between 0.75:2.25 and 1.25:2.25, or about1:2.

In another embodiment, the composition consists of

a. a polymeric biguanide in an amount of 750 to 1250 ppm, or about 1000ppm,

b. ion chelating agent in an amount of 1750 to 2250 ppm, or about 2000ppm, and

g. a solvent up to 100 wt %%, and optionally

e. a humectant in an amount of 40000 to 100000 ppm, or about 50000 or85000 ppm.

The composition of the present invention is believed to provide asynergistic interaction between a polymeric biguanide and a metal ionchelating agent to further enhance its antibacterial, cleansing effectand removal of the slough and necrotic tissue from a wound.

In one embodiment, the composition consists of

a. a polymeric biguanide,

b. a metal ion chelating agent,

c. a quaternary ammonium salt, and

g. a solvent

wherein the ratio of a:b:c is between 0.75:2.25:0.30 and 1.25:2.25:1.25,or about 1:2:1.

In another embodiment, the composition consists of

a. a polymeric biguanide in an amount of 750 to 1250 ppm, or about 1000ppm,

b. ion chelating agent in an amount of 1750 to 2250 ppm, or about 2000ppm,

c. a quaternary ammonium salt in an amount of 400 to 1250 ppm, or about500 or 1000 ppm, and

g. a solvent up to 100 wt %%, and optionally

e. a humectant in an amount of 40000 to 100000 ppm, or about 50000 or85000 ppm.

The composition of the present invention is believed to provideadditional synergistic interaction between a polymeric biguanide, and ametal ion chelating agent through the addition of a quaternary ammoniumsalt to further enhance its antibacterial, cleansing effect and removalof the slough and necrotic tissue from a wound. This allows for use ofthe ingredients in low doses with improved healing, even for long termtreatment and without toxic effects on the cells of the tissue to betreated.

In one embodiment, the composition consists of

a. a polymeric biguanide,

b. a metal ion chelating agent,

d. at least one surfactant, and

g. a solvent

wherein the ratio of a:b:d is between 0.75:2.25:0.75 and 1.25:2.25:2.25,or about 1:2:2.

In another embodiment, the composition consists of

a. a polymeric biguanide in an amount of 750 to 1250 ppm, or about 1000ppm,

b. ion chelating agent in an amount of 1750 to 2250 ppm, or about 2000ppm,

d. at least one surfactant in an amount of 1750 to 2250 ppm, or about2000 ppm, and

g. a solvent up to 100 wt %, and optionally

e. a humectant in an amount of 40000 to 100000 ppm, or about 50000 or85000 ppm.

In a further embodiment, the composition consist of

a. polyhexamethylene biguanide, in an amount of 750 to 1250 ppm, orabout 1000 ppm,

b. EDTA in an amount of 1750 to 2250 ppm, or about 2000 ppm,

d1 polysorbate 60 in an amount of 750 to 1250 ppm, or about 1000 ppm,

d2. undecylenamidopropyl betaine in an amount of 750 to 1250 ppm, orabout 1000 ppm,

g. water up to 100 wt %, and

e. glycerin in an amount of 40000 to 100000 ppm, or about 50000 or 85000ppm.

The composition of the present invention is believed to provideadditional synergistic interaction between a polymeric biguanide, and ametal ion chelating agent through the addition of at least onesurfactant to further enhance its antibacterial, cleansing effect andremoval of the slough and necrotic tissue from a wound. This allows foruse of the ingredients in low doses with improved healing, even for longterm treatment and without toxic effects on the cells of the tissue tobe treated.

In one embodiment, the composition consists of

a. a polymeric biguanide,

b. a metal ion chelating agent,

c. a quaternary ammonium salt,

d. at least one surfactant; and

g. a solvent

wherein the ratio of a:b:c:d is between 0.75:2.25:0.30:0.75 and1.25:2.25:1.25:2.25, or about 1:2:1:2.

In another embodiment, the composition consists of

a. a polymeric biguanide in an amount of 750 to 1250 ppm, or about 1000ppm,

b. ion chelating agent in an amount of 1750 to 2250 ppm, or about 2000ppm,

c. a quaternary ammonium salt in an amount of 400 to 1250 ppm, or about500 or 1000 ppm,

d. at least one surfactant in an amount of 1750 to 2250 ppm, or about2000 ppm, and

g. a solvent up to 100 wt %, and optionally

e. a humectant in an amount of 40000 to 100000 ppm, or about 50000 or85000 ppm.

In a further embodiment, the composition consist of

a. polyhexamethylene biguanide in an amount of 750 to 1250 ppm, or about1000 ppm,

b. EDTA in an amount of 1750 to 2250 ppm, or about 2000 ppm,

c. benzalkonium chloride in an amount of 750 to 1250 ppm, or about 1000ppm,

d1 polysorbate 60 in an amount of 750 to 1250 ppm, or about 1000 ppm,

d2. undecylenamidopropyl betaine in an amount of 750 to 1250 ppm, orabout 1000 ppm,

g. water up to 100 wt %, and

e. glycerin in an amount of 40000 to 100000 ppm, or about 50000 or 85000ppm.

The composition of the present invention is believed to provideadditional synergistic interaction between a polymeric biguanide, ametal ion chelating agent and a quaternary ammonium salt through addingat least one non-ionic surfactant to further enhance its antibacterial,cleansing effect and removal of the slough and necrotic tissue from awound.

The composition of the present invention is believed to provideadditional synergistic interaction between a polymeric biguanide, ametal ion chelating agent and a quaternary ammonium salt through addingat least one amphoteric surfactant to further enhance its antibacterial,cleansing effect and removal of the slough and necrotic tissue from awound.

In another embodiment, the at least one surfactant includes at least onenon-ionic surfactant or at least one amphoteric surfactant or mixturesthereof, which surfactants have a hydrophilic-lipophilic balance valuebetween 8 and 20, or between 10 and 18. In one embodiment, the non-ionicsurfactant may be Polysorbate or Polysorbate 60.

In a further embodiment, the amphoteric surfactant may beundecylenamidopropyl betaine or cocobetaine.

In a further embodiment, the composition consists of

a. a polymeric biguanide in an amount of 750 to 1250 ppm, or about 1000ppm,

b. ion chelating agent in an amount of 1750 to 2250 ppm, or about 2000ppm, and

g. a solvent up to 100 wt %,

and optionally one or more of the following ingredients

c. a quaternary ammonium salt in an amount of 400 to 1250 ppm, or about500 or 1000 ppm,

d. at least one surfactant in an amount of 1750 to 2250 ppm, or about2000 ppm,

e. a humectant in an amount of 40000 to 100000 ppm, or about 50000 or85000 ppm, and

f. a gelling agent in an amount of 1.5 to 2.5 wt %, or about 1.8 wt %,

In yet a further embodiment, the composition consists of

a. a polymeric biguanide in an amount of 750 to 1250 ppm, or about 1000ppm,

b. ion chelating agent in an amount of 1750 to 2250 ppm, or about 2000ppm,

d. at least one surfactant in an amount of 1750 to 2250 ppm, or about2000 ppm,

e. a humectant in an amount of 40000 to 100000 ppm, or about 50000 or85000 ppm, and

g. a solvent up to 100 wt %,

and optionally one or more of the following ingredients

c. a quaternary ammonium salt in an amount of 400 to 1250 ppm, or about500 or 1000 ppm, and

f. a gelling agent in an amount of 1750 to 2250 ppm, or about 2000 ppm,

The polymeric biguanide as used in any of the compositions outlinedabove may be polyhexamethylene biguanide (PHMB).

The ion chelating agent as used in any of the compositions outlinedabove may be ethylenediaminetetraacetic acid (EDTA).

The quaternary ammonium salt as used in any of the compositions outlinedabove may be benzalkonium chloride.

The at least one surfactant as used in any of the compositions outlinedabove may be Polysorbate or Polysorbate 60, or undecylenamidopropylbetaine or cocobetaine.

The humectant as used in any of the compositions outlined above may beglycerine.

The gelling agent as used in any of the compositions outlined above maybe hydroxyethyl cellulose.

The solvent as used in any of the compositions outlined above may bewater.

The pH in any of the compositions outlined above may be between 4 and 8,or 5 and 7, or 5.5 and 6.5.

The hydrophilic-lipophilic balance value in any of the compositionsoutlined above may be between 8 and 20 or between 10 and 18.

In an embodiment, the composition consists of

a. polyhexamethylene biguanide in an amount of 750 to 1250 ppm, or about1000 ppm,

b. ethylenediaminetetraacetic acid in an amount of 1750 to 2250 ppm, orabout 2000 ppm,

d1. one surfactant selected from Polysorbate or Polysorbate 60, in anamount of 750 to 1250 ppm, or about 1000 ppm,

d2. one surfactant selected from undecylenamidopropyl betaine orcocobetaine, in an amount of 750 to 1250 ppm, or about 1000 ppm,

e. glycerine in an amount of 40000 to 100000 ppm, or about 50000 or85000 ppm, and

g. water up to 100 wt %, and optionally

f. hydroxyethyl cellulose in an amount of 1.5 to 2.5 wt %, or about 1.8wt %, and

c. benzalkonium chloride in an amount of 400 to 1250 ppm, or about 500or 1000 ppm,

wherein the pH of the composition is between 4 and 8, or 5 and 7, or 5.5and 6.5.

The antimicrobial, cleansing composition according to the presentinvention pertains to eliminate microorganisms including, but notlimited to, gram negative and gram positive bacteria, virus, yeast,fungi, protozoa in wounds. The antimicrobial, cleansing compositionaccording to the present invention is a polymeric biguanide basecomposition that shows similar negligible or no cellular toxicity asmedical honey. The composition has no bacterial resistance and can thusbe used with improves efficacy against multidrug-resistant organisms,which may infect wounds and lead to increased morbidity, including themethicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.The composition accelerates wound healing, especially during long termtreatment due to the negligible cell toxicity of the composition.

The new composition can be used for wound healing in a mammal, such as ahuman, especially for removal of a biofilm. The wounds may be presentinside the mammal body or be present on the skin of the mammal. Lowdoses of the ingredients as present in the composition allow the use ofthe composition with minimal or no cellular toxicity for the mammalcells. This improves wound healing. The new composition thus improvessafety, efficacy and efficiency of wound healing compared to knowncompositions.

Due to the negligible or low cell toxicity of the composition, thecomposition is useful for long term treatment of a wound, such astreatment for more than two or five or ten or fourteen or twenty one, ormore days.

The antimicrobial, cleansing composition according to the presentinvention pertains for treatment of wounds containing soft-tissue and/orbones. The composition can be used as wound irrigation solutions eithermanually or automatically using Negative Pressure Wound Therapy devices.The composition can be made in form of ointments, creams, gels,solutions and wound dressings.

The composition of the invention may be a pharmaceutical composition.

The inventions relates to the composition as defined herein for use as amedicament. The composition as defined herein, can be used in thetreatment, prevention or reduction of a disease, disorder or conditionrelated to microbials, with reduced or without cellular toxicity for thecells. The composition as defined herein, can be used in the treatment,prevention or reduction of infected or inflamed wounds in a mammal. Theinfection or inflammation may be acute or chronic. The mammal may be ahuman or an animal, such as a rat. The composition as defined herein,can be used for debridement of wounds and for removal of necrotictissue. The inventions relates to the composition as defined herein foruse in a treatment, prevention or reduction of a disease, disorder orcondition selected from the group comprising or consisting of acute orchronic infection, acute or chronic inflammation, such as gingivitis,diabetic ulcers, second-degree partial-thickness burns, traumaticwounds, Acanthamoeba keratitis, etc.

The invention relates to a use of the composition as defined herein in atreatment, prevention or reduction of a disease, disorder or conditionrelated to microbials. The invention relates also to a use of thecomposition as defined herein for the treatment, prevention or reductionof growth of microbials, especially in wounds present in or on a mammal.The invention especially relates to a use of the composition as definedherein for a treatment or prevention of biofilms or the reduction offormation of biofilms, such as biofilms in the middle ear. Said biofilmsbeing produced by the microbials. The invention further relates to a useof the composition as defined herein for debridement of wounds and forremoval of necrotic tissue and/or improvement of wound healing. Further,the invention relates to a use of the composition as defined herein fora treatment, prevention or reduction of infected bones and/or infectedcavities in a mammal body.

The invention also relates to topical administration of the compositionas defined herein for use in a treatment, prevention or reduction of adisease, disorder or condition related to microbials, or treatment,prevention or reduction of a disease, disorder or condition selectedfrom the group comprising or consisting of acute or chronic infection,acute or chronic inflammation, such as gingivitis, diabetic ulcers,second-degree partial-thickness burns, traumatic wounds, Acanthamoebakeratitis, necrotizing fasciitis, venous stasis disease, pressureulcerations, such as leg ulcers, or carcinomas, or treatment, preventionor reduction of acute or chronic infected wounds, or acute or chronicinflamed wounds, or formation of biofilms, or for use in debridement ofwounds and for removal of necrotic tissue, in a mammal or any of theuses mentioned above. Said disease, disorder or condition may be atopical infection.

The composition can even be used for disinfection of surfaces, such asbedding, surgery tables, tubing, and reusable medical equipment. Thecomposition can be used for wound cleansing solutions, wound dressings,contact lens solutions, mouth washes solution, cosmetic preservative,food disinfectant, veterinary applications, swimming pool cleaners, andin industrial water treatment.

The composition may be used against Pseudomonas aeruginosa,Staphylococcus aureus (also the methicillin-resistant type, MRSA),Escherichia Coli, Candida albicans (yeast), Aspergillus brasiliensis(mold), vancomycin-resistant enterococci, and Klebsiella pneumoniae(carbapenem-resistant enterobacteriaceae).

The invention further relates to a method of treating, preventing orreducing a disease, disorder or condition related to microbials, whichcomprises administering to a mammal in need thereof a therapeuticallyeffective amount the composition as defined herein. In one embodiment,the method relates to treatment, prevention or reduction of a disease,disorder or condition selected from the group comprising or consistingof acute or chronic infection, acute or chronic inflammation, such asgingivitis, diabetic ulcers, second-degree partial-thickness burns,traumatic wounds, Acanthamoeba keratitis, necrotizing fasciitis, venousstasis disease, pressure ulcerations, such as leg ulcers, or carcinomas,or treatment, prevention or reduction of acute or chronic infectedwounds, or acute or chronic inflamed wounds, or formation of biofilms,or for use in debridement of wounds and for removal of necrotic tissue.

The invention also relates to the use of the composition as definedherein, in the manufacture of a medicament for the treatment, preventionor reduction of a disease, disorder or condition related to microbials,or treatment, prevention or reduction of a disease, disorder orcondition selected from the group comprising or consisting of acute orchronic infection, acute or chronic inflammation, such as gingivitis,diabetic ulcers, second-degree partial-thickness burns, traumaticwounds, Acanthamoeba keratitis, necrotizing fasciitis, venous stasisdisease, pressure ulcerations, such as leg ulcers, or carcinomas, ortreatment, prevention or reduction of acute or chronic infected wounds,or acute or chronic inflamed wounds, or formation of biofilms, or foruse in debridement of wounds and for removal of necrotic tissue, in amammal.

The present invention also relates to a system adapted for use in woundhealing comprising a negative pressure source, a canister for collectingwound exudate, a reservoir containing the composition as defined above,a liquid injector device for transferring a predetermined volume of thecomposition and connector tubes to transfer the composition to and froman infected soft tissue or bone, a negative pressure wound dressing,which system is adapted to draw fluid from the reservoir to the wounddressing and draw fluid away from the wound dressing after a holdingtime of 5 to 60 minutes.

The wound dressing may be soaked with the composition of the invention.Adhesive film or tape may be used to stabilize the wound dressing andtubes.

In one embodiment, the system also comprises a control unit adapted forcontrolling the flow of composition to and from the wound. In a furtherembodiment, the control unit is adapted to start the negative pressuresource after a holding time of between 5 and 60 minutes, so that thecomposition is removed from the wound and fresh composition is added tothe wound. The system may include valves to further control the flow ofcomposition through the tubes.

The invention also relates to a use of the system as defined above foruse in a treatment, prevention or reduction of a disease, disorder orcondition related to microbials, or treatment, prevention or reductionof a disease, disorder or condition selected from the group comprisingor consisting of acute or chronic infection, acute or chronicinflammation, such as gingivitis, diabetic ulcers, second-degreepartial-thickness burns, traumatic wounds, Acanthamoeba keratitis,necrotizing fasciitis, venous stasis disease, pressure ulcerations, suchas leg ulcers, or carcinomas, or treatment, prevention or reduction ofacute or chronic infected wounds, or acute or chronic inflamed wounds,or formation of biofilms, or for use in debridement of wounds and forremoval of necrotic tissue, in a mammal, such as a rat, or any of theuses mentioned above.

An advantage of the system according to the invention is a continuous(periodical) flow of the composition to and from the wound. Thisprevents biofilm formation, improves efficacy and efficiency of woundhealing. The system is especially useful for long term treatment of awound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a negative pressure system adapted for use in woundhealing.

FIG. 2 shows A (X100), B (X400); Histopathologic changes in the ulcersof Group PHMB on day 7 showed ulceration, chronic inflammation, andgranulation tissue formation, and C (X100), D (X400); Histopathologicchanges in the lesions of the same group on day 21 showed fullre-epithelialization and neovascularization.

DETAILED DESCRIPTION Definitions

A wound may be defined as a breakdown in the protective function of theskin, the loss of continuity of epithelium, with or without loss ofunderlying connective tissue (i.e. muscle, bone, nerves), due to injuryto the skin or underlying tissues/organs caused by, but not limited to,a trauma, burns, diabetic ulcers, severe infections, such as necrotizingfasciitis, venous stasis disease, and pressure ulcerations or as aresult of disease, such as leg ulcers or carcinomas.

A wound may be defined as a closed cavity with existing pathology asinfection, inflammation (open abdominal wound, abscess, infected jointwith or without implant), or a cavity which is connected to externalenvironment by a fistula.

Medical honey or (MEDIHONEY® Comvita Ltd, New Zealand) is an advancedwound care dressing used for its clinical ability to promote the removalof necrotic tissue and advance wound healing. Cellular toxicity of thecomposition of the present invention is compared to Medihoney through anexperimental animal study.

“Biofilm” may be defined as colonization with microorganisms aspolymicrobial communities encased within a matrix of extracellularpolymeric substance (EPS). This community of microorganisms are attachedto each other, often in conjunction with a surface, and form communityof microorganisms. The combination of a community of microorganisms,encased within self-generated EPS and attached onto a surface (liquid orsolid) may be defined as a biofilm.

“A” and “the” are understood to include not just one, but one or more ofthe subject following the “a” or “the”. Thus, a surfactant is understoodto means one or more surfactants.

“wt %” is weight percentages of the total weight of the composition.

“ppm” is parts per million.

Microbials may be gram negative and gram positive bacteria, virus,yeast, fungi, protozoa, and the like.

The present invention relates to a wound healing formula comprising atleast one anti-microbial polymeric biguanide and at least one metal ionchelating agent, with or without one antimicrobial quaternary ammoniumcompounds.

Combination of a polymeric biguanide with a metal ion chelating agentand optionally a quaternary ammonium salt may give a synergy effectbetween these elements at low concentration in order to provide aneffective biocides composition. This invention is based not only onproviding a composition that shows excellent biocide effect, but alsodemonstrates a negligible or low cellular toxicity. The combination ofactive ingedients in low concentration, results in a mixture with lowcellular toxicity and better compatibility with the environment.

The term “long term treatment” has the meaning of a treatment for morethan two or five or ten or fourteen or twenty one, or more days.

Polymeric Biguanide

A preferred polymeric biguanide is polyhexamethylene biguanide (PHMB)commercially available from e.g. Arch Chemicals (CAS No 51274-09-0).

It is well known that polyhexamethylene biguanide (PHMB) has broadspectrum bactericidal and fungicidal action with good tissue tolerance.PHMB has been used in wound cleansing solutions, wound dressings,contact lens solutions, mouth washes solution, surface disinfectant,cosmetic preservative, food disinfectant, veterinary applications,swimming pool cleaners, and in industrial water treatment.

PHMB has been shown to be effective against Pseudomonas aeruginosa,Staphylococcus aureus (also the methicillin-resistant type, MRSA),Escherichia Coli, Candida albicans (yeast), Aspergillus brasiliensis(mold), vancomycin-resistant enterococci, and Klebsiella pneumoniae(carbapenem-resistant enterobacteriaceae).

Tests have been performed on S. aureus to investigate the potentialrisks of this bacterium developing resistance to PHMB. The risk wasfound to be very low. An additional positive influence on theinflammatory process of wound healing, especially in infected orcritically colonized wounds has been the binding of inflammatoryparameters such as free radicals, showing its antioxidative potential.The clinical effect of using PHMB in some non-healing wounds has beenpromising.

Cationic Quaternary Ammonium Compounds

The composition in this invention may comprises a cationic quaternaryammonium compound, which is selected from a group of quaternary ammoniumsalts with long alkyl chains, such as benzalkonium chloride,benzethonium chloride, methylbenzethonium chloride, cetalkoniumchloride, cetylpyridinium chloride, cetrimonium, cetrimide, dofaniumchloride, tetraethylammonium bromide, didecyldimethylammonium chlorideand domiphen bromide, and mixtures thereof. Preferred used quaternaryammonium compounds are benzethonium chloride, methylbenzethoniumchloride, and most preferable is benzalkonium chloride with molecularformula C21H38C1N and CAS No. 8001-54-5.

Quaternary ammonium compounds are cationic detergents, as well asdisinfectants and surfactants, and as such can be used to remove organicmaterial. Quaternary ammonium compounds are deactivated by anionicdetergents. Certain quaternary ammonium compounds, especially thosecontaining long alkyl chains, are used as antimicrobials anddisinfectants.

Quaternary ammonium compounds are believed to act by disrupting the cellmembrane and are also good against fungi, amoebas, and envelopedviruses. Quaternary ammonium compounds are lethal to a wide variety oforganisms except endospores, Mycobacterium tuberculosis andnon-enveloped viruses.

Metal Ion Chelating Agents

Suitable chelating agents comprise, but are not limited to, mixed saltsof Ethylenediaminetetraacetic acid (EDTA), such as disodium, trisodium,tetrasodium, dipotassium, tripotassium, tetrapotassium, lithium,dilithium, ammonium, diammonium, triammonium, tetraammonium, calcium andcalcium disodium, more preferably disodium, trisodium or tetrasodiumsalts of EDTA, preferably disodium EDTA and tetrasodium EDTA.

EDTA is used in the present invention for its antibiofilm effect. EDTAis a well-known metal chelating agent. EDTA has been used as apermeating and sensitizing agent for treating biofilm-associatedconditions in dentistry, on medical devices, and in veterinary and humanmedicine. The effect of EDTA and its ability to chelate and potentiatethe cell walls of bacteria and its ability to destabilize a biofilm bysequestering calcium, magnesium, zinc and iron makes it a suitable agentfor use in the prevention and management of biofilms.

EDTA has been utilized for the control of microorganisms and biofilmsoften by being combined with other actives including alcohol,antibiotics, citric acid, polyhexamethylene biguanide (PHMB), quaternaryammonium compounds, silver, iodine, surfactants and other antiseptics.

Surfactants

The antimicrobial composition in the invention for debridement ofwounds, may include at least one non-ionic surfactant or at least oneamphoteric surfactant or mixtures thereof.

The surfactants can have an HLB (hydrophilic-lipophilic balance) valueof 5-25, or 8-20, or 10-18 in order to maintain the biocidal activity ofthe antimicrobial agents.

The term surfactant comes from the words surface active agent.Surfactants are one of many different compounds that make up adetergent. They are added to remove dirt from skin, clothes andhousehold articles. Surfactants have a hydrophobic part and ahydrophilic part. The hydrophobic part consists of an unchargedcarbohydrate group that can be straight, branched, cyclic or aromatic.Dependent on the nature of the hydrophilic part, the surfactants areclassified as an-ionic, non-ionic, cationic or amphoteric.

Nonionic Surfactants

A surfactant with a non-charged hydrophilic part, e.g. ethoxylate, isnon-ionic. These substances are well suited for cleaning purposes. Theyhave a wide application within cleaning detergents and include groupslike fatty alcohol polyglycosides, alcohol ethoxylates etc.

Suitable non-ionic surfactants include Polysorbate 60 with the chemicalformula C24H46O6, Registry number CAS 1338-41-6, decyl glucoside, andlauryl glucoside polyethylene glycol esters of fatty acids, e.g.,coconut, polysorbate, polyoxyethylene.

Other examples may be Polysorbate 20 available under the trademarkTween® 20, polyoxyethylene (40) stearate available under the trademarkMyrj® S40, polyoxyethylene (25) propylene glycol stearate availableunder the trademark Atlas® G 2612, and polyoxyethylene (23) lauryl etheravailable under the trade-mark Brij® (35).

Other non-ionic surfactants can be added, such as theethyleneoxide/propylene oxide block copolymers of poloxamers, andpoloxamers. Poloxamers and poloxamines are preferred, and poloxamers aremost preferred. Poloxamers and poloxamines are available from BASF Corp.under the trademarks of Pluronic® and Tetronic®.

The present invention pertains to the use of polysorbate that haveamphiphilic properties and are non-ionic, in particular Polysorbate 60in combination with PHMB for the treatment of persons and animalssuffering from acute or chronic wounds, for example, in woundantiseptics, as wound irrigation solutions manually or as woundirrigation solutions automatically using Negative Pressure Wound Therapydevices, in ointments, creams, gels, solutions and wound dressings.

Amphoteric Surfactants

Amphoteric surfactants for use in the composition in this invention canbe selected from a group of betaine, amine oxide and imidazoliumderivatives. Of said surfactants, are particularly preferred alkylbetaines, alkyl amine oxides and alkyl imidazolium derivatives.

Preferred alkyl betaines include Lauryldimethylbetaine (Cas No:683-10-3), Cocobetaine (Cas No: 68424-94-2), Myristyl betaine (Cas No:2601-33-4), Decyl betaine (Cas No: 2644-45-3), Lauryl sultaine (Cas No:14933-08-5), Oleyldimethylbetaine (Cas No: 871-37-4), Dodecylbetaine(Cas No: 55142-08-0), Caprylyl betaine (Cas No: 27593-14-2) and Behenylbetaine (Cas No: 26920-62-7).

Preferred amine oxides include Lauramine oxide (Cas No: 1643-20-5),Lauramidopropylamine oxide (Cas No: 61792-31-2), Cocamine oxide (Cas No:61788-90-7), Cocamidopropylamine Oxide (Cas No: 68155-09-9), Myristamineoxide (Cas No: 3332-27-2) and Soyamidopropylamine Oxide (Cas No:223707-70-8).

Preferred imidazoline surfactants include Disodium cocoamphodiacetate(Cas No: 68650-39-5), Di sodium lauroamphodiacetate (Cas No:14350-97-1), Sodium lauroamphoacetate (Cas No: 26837-33-2), Disodiumcocoamphodipropionate (Cas No: 68604-71-7) and Sodium cocoamphoacetate(Cas No: 68334-21-4).

For the amphoteric surfactants, the charge of the hydrophilic part iscontrolled by the pH of the solution. This means that they can act asanionic surfactant in an alkalic solution or as cationic surfactant inan acidic solution.

Anionic Surfactants

When the hydrophilic part of the surfactant consists of a negativelycharged group like alkyl sulphonate, alkyl sulphate or carboxylate oralkyl benzene sulphonates. Anionic surfactants neutralized the biocideseffect of PHMB and therefor the combination of these two is notdesirable. For this reason, the antimicrobial composition in thisinvention does not include anionic surfactants.

Humectant

The antimicrobial formula comprising at least one humectant. A driedwound cannot be healed. Humectants are used in formulations to increasethe moisture content of the skin. Humectants promote water retentionwithin the stratum corneum and prevent wounds from drying.

Humectants can be selected from a group comprising glycerine, lecithin,dipropylene glycol, polyethylene glycol and its derivate, 1,2-propyleneglycol, 1,3 butylene glycol, and 1,2,6-hexanetriol, beta-sitosterol,inositol, glyceryl stearate, panthenol, and xylitol.

Solvent

The solvent may be selected from the group comprising Lactate Ringersolution, Ringer solution without lactate, Normal saline and purifiedwater as a solvent. In the present invention purified water bydistillation is preferably selected a solvent.

Buffer

The composition has preferably a pH between 4.0 and 8, in particularbetween 5 and 7 and preferably between 5.5 and 6.5. The role of woundbed pH is significant during the healing of wounds, and prolongedacidification of the wound bed has been shown to increase the healingrate in chronic venous leg ulcers. Suitable buffers to adjust pH caninclude sodium citrate, potassium citrate, and citric acid. Anotherpreferred buffer for pH adjustment is a combination of disodiumethylenediaminetetraacetic acid salt and trisodiumethylenediaminetetraacetic acid salt.

Gelling Agent

A gelling agent is a substance which can increase the viscosity of aliquid without substantially changing other properties. However, anycellulose derivative (e.g. methyl cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, hydroxyethyl cellulose) may beused in order to get a gel form of the composition either alone or incombination with other gelling agents. Another preferred gelling agentis carboxypolymethylene, available under the trade mark Carbopol®.Carbopol® 934P type is a high purified product and can be used in theformulation of the present invention.

In a preferred aspect, the aqueous antimicrobial compositions describedherein include biocidal polymeric biguanides at a concentration rangingfrom 100 ppm to 10,000 ppm, or ranging from 300 ppm to 5,000 ppm, orranging from 1,000 ppm to 2,000 ppm, or ranging from 750 ppm to 1,500ppm, or ranging from 750 ppm to 1,250 ppm, or ranging from 850 ppm to1,150 ppm.

When quaternary ammonium compound is present, it is preferably presentin an amount in the range 50 ppm to 10,000 ppm in the diluted form, morepreferably in an amount in the range 500 ppm to 5,000 ppm, morepreferably in an amount in the range 1,000 to 2,000 ppm, or ranging from750 ppm to 1,500 ppm, or ranging from 750 ppm to 1,250 ppm, or rangingfrom 850 ppm to 1,150 ppm

The amount of metal ion chelating agents is at least 200 ppm. Apreferred concentration ranging is from 300 ppm to 10,000 ppm, orranging from 750 ppm to 5,000 ppm, or ranging from 1,000 to 3,000 ppm,or ranging from 1500 ppm to 2,500 ppm, or ranging from 1750 ppm to 2,250ppm, or ranging from 1850 ppm to 2,150 ppm. EDTA would provideantibiofilm ability by chelating calcium and magnesium ions, whichmaintain the structure of the biofilm, and remove iron which is vital tomicrobial virulence and pathogenicity.

In a preferred aspect, the formulation is a solution comprising at leastone non-ionic or at least one amphoteric surfactant or mixtures thereofat a concentration of from 100 ppm to 10,000 ppm in the diluted form, orranging from 500 ppm to 5,000, or ranging from 2,000 ppm to 4,000 ppm,or ranging from 1500 ppm to 2,500 ppm, or ranging from 1750 ppm to 2,250ppm, or ranging from 1850 ppm to 2,150 ppm.

The amount of humectant agent is at least 5,000 ppm. A preferredconcentration ranging is from 70,000 to 100,000 ppm, or ranging from30,000 ppm to 100,000 ppm. More preferably 40,000 ppm to 60,000 ppm or75,000 ppm to 90,000 ppm.

The formulation for gels consists of 0.05-10% gelling agent by weight,or 0.5-5 wt %, or 1-3 wt %. or 1.5-2 wt % and completed to 100% byadding solvent.

In topical applications, the antimicrobial composition may be preparedin different forms, such as, but not limited to, liquids, creams, foams,lotions, gels and aerosols. The composition of the invention can beadministered by using wound dressing materials and non-woven, foams,sponges, swabs, surgical gauze pad, cloth. The materials may besaturated or soaked in any one of the composition of the invention, asdefined above.

In topical application, the antimicrobial composition product may alsobe administered to wounds or body cavities in liquid form for manuallyor instillation or automatically irrigation in conjunction with NegativePressure Wound Therapy devices.

The liquid flow system and wound dressing utilize a Negative PressureWound Therapy System including a negative pressure source device andnegative pressure wound dressing and connecting tubes.

The liquid flow system may draw liquid form of said composition bygravity feed system. The liquid flow system may draw liquid form of saidcomposition by using an electrical or mechanical pump.

FIG. 1 shows a system or liquid flow comprising a negative pressuresource N10, a canister (C10) for collecting wound exudate, a reservoir(R10) containing the composition (F) as defined above, a liquid injectordevice (I10) for transferring a (predetermined) volume of thecomposition and connector tubes (T10, T20, T30) to transfer thecomposition to and from an infected soft tissue or bone (S10). A wounddressing (W10) may be used that can be soaked with the composition ofthe invention. Adhesive film or tape may be used to stabilize the wounddressing and tubes.

The system may also comprises a control unit (not shown) adapted forcontrolling the flow of composition to and from the wound. The controlunit may be adapted to start the negative pressure source after aholding time of between 5 and 60 minutes, so that the composition isremoved from the wound and fresh composition is added to the wound. Thesystem may include valves positioned on the tubes or the reservoir,canister, etc. to further control the flow of composition through thetubes.

The system may utilize the control unit, which may be a computerizesystem, to “start” and “stop” to control delivery of a certain volume ofthe said composition to a wound dressing.

The system may after a “holding time” start the negative pressure deviceto draw the said composition away from the wound dressing.

Said holding time is about 5 to 60 minutes, preferably 10 to 30 minutes,and most preferably 20 minutes in order for the composition to have anantibacterial and cleansing effect.

Formulations, especially topical formulations of the antimicrobialcomposition may additionally comprise diluents, excipients and otheradditives commonly used in pharmaceutical formulations, such as forexample emulsifiers, gelling agents, moisturizers, stabilizers, timerelease agents.

Examples on Formulation

Compositions according to the present invention include a biguanide anda chelating agent. The composition may further include other ingredientsas outlined above. The following compositions have been prepared;

EXAMPLE 1

Agents Amount (ppm) 1) PHMB 1000 2) EDTA 2000 3) Glycerin, humectant50000 4) Polysorbate 60 1000 5) Cocobetaine 1000 6) Purified water To100%

EXAMPLE 2

Agents Amount 1) PHMB 1000 2) EDTA 2000 3) Glycerin 50000 4) Polysorbate60 1000 5) Undecylenamidopropylbetainesurfactants 1000 6) Purified waterTo 100%

EXAMPLE 3

Agents Amount (ppm) 1) PHMB 1000 2) Benzalkoniumchloride 1000 3) EDTA2000 4) Glycerin 50000 5) Polysorbate 60 1000 6) Cocobetaine 1000 7)Purified water To 100%

EXAMPLE 4

Agents Amount (ppm) 1) PHMB 1000 2) Benzalkonium chloride 1000 3) EDTA2000 4) Glycerin 50000 5) Polysorbate 60 1000 6) Undecylenamidopropylbetaine 1000 7) Purified water To 100%

EXAMPLE 5

Agents Amount (ppm) 1) PHMB 1000 2) EDTA 2000 3) Glycerin 85000 4)Polysorbate 60 1000 5) Cocobetaine 1000 6) Hydroxyethyl cellulose 1.8wt. % 7) Purified water To 100%

EXAMPLE 6

Agents Amount (ppm) 1) PHMB 1000 2) EDTA 2000 3) Glycerin 50000 4)Polysorbate 60 1000 5) Undecylenamidopropyl betaine 1000 6) Hydroxyethylcellulose 1.8 wt. % 7) Purified water To 100%

EXAMPLE 7

Agents Amount (ppm) 1) PHMB 1000 2) Benzalkonium chloride 500 3) EDTA2000 4) Glycerin 85000 5) Polysorbate 60 1000 6) Cocobetaine 1000 7)Hydroxyethyl cellulose 1.8 wt. % 8) Purified water To 100%

EXAMPLE 8

Agents Amount (ppm) 1) PHMB 1000 2) Benzalkonium chloride 500 3) EDTA2000 4) Glycerin 85000 5) Polysorbate 60 1000 6) Undecylenamidopropylbetaine 1000 7) Hydroxyethyl cellulose 1.8 wt. % 8) Purified water To100%

Experimental Animal Study Experiment 1 Animals and Excisional WoundModel

This work followed the guidelines of National Institute of Health forthe Care and Use of Laboratory Animals. Twenty-two healthy adult maleSprague-Dawley rats (mean weight 350 g) were selected for thisexperimental work. The rats were kept in clean and separate wire bottomcages, and housed in temperature-controlled (22±2° C.) andhumidity-controlled (55±15%) rooms with 12-hour light: dark photocycles. All the rats had free access to equal amounts of standard animalfood and water. The rats were matched their environment for one weekbefore starting the study.

Before wounding, the rats were anaesthetized with an intramuscularinjection of thiopental sodium (40 mg/kg; Biochemie, GmbH, Austria) andxylazine (10 mg/kg; Alfasan International, Woerden, Netherlands); then,after the back hair was shaved, the site of wound creation wasdisinfected with alcohol ethylic solution. Next, a full-thicknessexcisional circular skin wound (diameter of 20 mm and depth of 2 mm) wascreated on the dorsum of each rat using scissors and forceps.

To conduct the research, the rats were randomly categorized into twogroups (n=11): (1): the group for which the composition according tothis invention (example 2) was applied on the wound surface area (group1); and (2): the group for which Medihoney®, Comvita Ltd, New Zealand,was applied on the wound surface area (group 2). During the studyperiod, the aforementioned products were applied daily to the woundswith a disposable applicator so that a thin layer that fully covered thewound surface area was created (no dressing was used in the experiment).Both groups were followed for 21 days.

Photographic Assessment of Wound Healing

The healing process was monitored photographically from images of eachskin wound taken with a 12.1 megapixel digital camera (PowerShot G9;Canon, Tokyo, Japan). To calibrate the magnification of the photographs,the camera was kept at a fixed distance of 10 cm from the wound surface(in a vertical view), and a fine-line ruler was placed at wound level atthe time of imaging. Photos were taken on days 0, 7, and 21post-wounding and analyzed using the Photoshop CS Program (AdobeSystems, San Jose, Calif., USA) (analysis menu; record measurementscommand). The results are shown in FIG. 2.

Histopathological Evaluation of Wound Healing

Semi-circular full-thickness skin biopsies from the wound sites of ratsin both groups were taken on days 7 (half of the wound site with amargin of 2 mm) and 21 (the remaining part with a 2 mm margin)post-wounding. The animals were anesthetized by inhalation of ether onday 7 and euthanized with ether on day 21.

Tissue samples were washed with sterile normal saline, immediately fixedin buffered formaldehyde (10% formalin), and sent for histopathologicalassessment using established techniques (haematoxylin and eosin andMasson-trichrome stainings, and light microscopic examination).

The scoring system developed by Abramov and his colleagues forhistopathological evaluation of the wound healing process was adapted tothis study. This scoring system uses the following features: the amountof acute and chronic inflammatory infiltrates, the amount and maturationof granulation tissue, neovascularization, collagen deposition, andreepithelialisation. All the investigators who assessed the tissuesamples or analysed the images were blinded to the agents given.

Statistical Analysis

The results are presented as mean±standard deviation (SD). Statisticalcomparisons were made using the Mann-Whitney U-test (SPSS Statisticssoftware, version 16; Chicago, Ill., USA). P values less than 0.05 wereconsidered statistically significant.

Results Wound Contraction

The mean±SD values of wound surface area were calculated for each of thegroups using the images taken on days 0, 7, and 21, post-wounding (Table1). According to the results, there was no statistically significantdifference in the mean wound surface area between the two groups in the0, 7^(th), and 21s^(t) days of experiment. The rats' wounds showed noapparent sign of infection during the study period.

TABLE 1 The mean ± SD of wound surface area (mm²) in two groups ondifferent days post-wounding. Day 0 7 21 Group 1 310.16 ± 13.22 48.54 ±11.34  0.0 ± 0.00 Group 2 316.87 ± 13.67 50.83 ± 9.49  0.26 ± 0.49P-value 0.123 0.234 0.187

Histopathological Examinations

The results of the histopathological assessment are summarized in Table2. According to this study, there was no statistically significantdifference between the two groups regarding the factors of acute andchronic inflammation, amount and maturation of granulation tissue,neovascularization, collagen deposition, and reepithelialisation. Nonecrosis was seen.

CONCLUSION

It has been shown that, daily topical application of the compositionaccording to the present invention (example 2), on fresh skin wounds hassimilar efficacy to Medical honey for the healing process.

In addition, no significant negative impact (i.e. cellular toxicity) onthe parameters that were involved in the wound healing process occurredfollowing the usage of the composition of the present invention (example2).

TABLE 2 Mean ± SD values of histopathological scores of wound healingamong the different study groups. Groups Day 7 Day 21 Parameter Group 1Group 2 P-Value Group 1 Group 2 P-Value Acute and 2.00 ± 1.90 ± 1.002.54 ± 1.81 ± .069 Chronic 0.89 0.94 0.52 0.87 inflammation Amount of2.54 ± 2.63 ± 0.60 0.72 ± 0.81 ± 0.34 granulation 0.52 0.50 1.00 0.75tissue Granulation 2.45 ± 2.54 ± 0.91 1.36 ± 2.09 ± 0.28 tissue 0.520.52 1.56 1.37 maturation Collagen 1.63 ± 2.00 ± 0.08 1.54 ± 2.00 ± 0.23deposition 0.67 0.44 0.52 0.89 Reepithelial- 0.45 ± 0.54 ± 0.51 2.72 ±2.72 ± 0.83 ization 1.03 1.03 0.64 0.64 Neovascular- 3.00 ± 3.00 ± 1.003.00 ± 3.00 ± 1.00 ization 0.00 0.00 0.00 0.00

1. An antimicrobial and cleansing composition comprising: a polymericbiguanide (ppm), a metal ion chelating agent (ppm), and a solvent (wt%), wherein the ratio of the polymeric biguanide to the metal ionchelating agent is between 0.75:2.25 and 1:2.
 2. The compositionaccording to claim 1, wherein the composition comprises: a polymericbiguanide (ppm), a metal ion chelating agent (ppm), a quaternaryammonium salt (ppm), and a solvent (wt %), wherein the ratio of thepolymeric biguanide to the metal ion chelating agent to the quaternaryammonium salt is between 0.75:2.25:0.30 and 1:2:1.
 3. The compositionaccording to claim 1, wherein the composition comprises: a polymericbiguanide (ppm), a metal ion chelating agent (ppm), at least onesurfactant (ppm), and a solvent (wt %), wherein the ratio of thepolymeric biguanide to the metal ion chelating agent to the at least onesurfactant is between 0.75:2.25:0.75 and 1:2:2.
 4. The compositionaccording to claim 1, wherein the composition comprises: a polymericbiguanide (ppm), a metal ion chelating agent (ppm), a quaternaryammonium salt (ppm), at least one surfactant (ppm), and a solvent (wt%), wherein the ratio of the polymeric biguanide to the metal ionchelating agent to the quaternary ammonium salt to the at least onesurfactant is between 0.75:2.25:0.30:0.75 and 1:2:1:2.
 5. Thecomposition according to claim 1, which further comprises of ahumectant.
 6. The composition according to claim 1, wherein thecomposition comprises: the polymeric biguanide in an amount of 750 to1250 ppm, or about 1000 ppm, the metal ion chelating agent in an amountof 1750 to 2250 ppm, or about 2000 ppm, and the solvent up to 100 wt %,and in addition optionally one or more of the following ingredients: aquaternary ammonium salt in an amount of 400 to 1250 ppm, or about 500or 1000 ppm, at least one surfactant in an amount of 1750 to 2250 ppm,or about 2000 ppm, a humectant in an amount of 40000 to 100000 ppm, orabout 50000 or 85000 ppm, and a gelling agent in an amount of 1.5 to 2.5wt %, or about 1.8 wt %.
 7. The composition according to claim 6,wherein the composition consists of polyhexamethylene biguanide in anamount of 750 to 1250 ppm, or about 1000 ppm, EDTA in an amount of 1750to 2250 ppm, or about 2000 ppm, polysorbate 60 in an amount of 750 to1250 ppm, or about 1000 ppm, undecylenamidopropyl betaine in an amountof 750 to 1250 ppm, or about 1000 ppm, water up to 100 wt %, andglycerin in an amount of 40000 to 100000 ppm, or about 50000 or 85000ppm.
 8. The composition according to claim 6, wherein the compositionconsists of polyhexamethylene biguanide in an amount of 750 to 1250 ppm,or about 1000 ppm, EDTA in an amount of 1750 to 2250 ppm, or about 2000ppm, benzalkonium chloride in an amount of 750 to 1250 ppm, or about1000 ppm, polysorbate 60 in an amount of 750 to 1250 ppm, or about 1000ppm, undecylenamidopropyl betaine in an amount of 750 to 1250 ppm, orabout 1000 ppm, water up to 100 wt %, and glycerin in an amount of 40000to 100000 ppm, or about 50000 or 85000 ppm.
 9. The composition accordingto claim 1, wherein the composition is a liquid at a pH from 5 to
 7. 10.The composition according to claim 3, wherein the at least onesurfactant includes at least one non-ionic surfactant or at least oneamphoteric surfactant or mixtures thereof, which surfactants have ahydrophilic-lipophilic balance value between 8 and 20, or between 10 and18.
 11. The composition according to claim 1, wherein the compositionfurther comprises one or more of the following ingredients: a quaternaryammonium salt, at least one surfactant, a humectant, and/or a gellingagent.
 12. The composition according to claim 1, wherein the compositionhas minimal or no cellular toxicity for mammal cells.
 13. A method oftreatment, prevention or reduction of a disease, disorder or conditionin a subject selected from the group consisting of acute or chronicinfection, acute or chronic inflammation, diabetic ulcers, second-degreepartial-thickness burns, traumatic wounds, Acanthamoeba keratitis,necrotizing fasciitis, venous stasis disease, pressure ulcerations, andcarcinomas, comprising administering a therapeutic effective amount of acomposition according to claim 1 to the subject.
 14. The method of claim13, wherein the disease, disorder, or condition is a topical infection.15. The method of claim 13, wherein the disease, disorder or conditionis acute or chronic infected wounds, or acute or chronic inflamedwounds.
 16. A liquid flow system for topical administration of thecomposition according to claim 1, whereby the system comprises anegative pressure source, a canister for collecting wound exudate, areservoir containing the composition, a liquid injector device fortransferring a predetermined volume of the composition; connector tubesto transfer the composition to and from an infected soft tissue or bone,and a negative pressure wound dressing, wherein the system is adapted todraw fluid from the reservoir to the wound dressing and draw fluid awayfrom the wound dressing after a holding time of 5 to 60 minutes.